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1.
Cell ; 180(2): 323-339.e19, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31928845

RESUMO

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.


Assuntos
Proteínas do Tecido Nervoso/ultraestrutura , Receptores de Peptídeos/metabolismo , Tenascina/metabolismo , Animais , Adesão Celular/fisiologia , Cristalografia por Raios X/métodos , Células HEK293 , Humanos , Células K562 , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL/embriologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/ultraestrutura , Ligação Proteica/fisiologia , Proteínas/metabolismo , Proteínas/ultraestrutura , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos/ultraestrutura , Sinapses/metabolismo , Tenascina/ultraestrutura
2.
Clin Nucl Med ; 45(3): 241-243, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31977472

RESUMO

We present here a case with ß-radiation-refractory metastatic neuroendocrine tumors, who demonstrated an excellent therapy response after 1 cycle of Ac-DOTATOC, without any significant adverse effects even after 10 cycles of ß-emitter peptide receptor radionuclide therapy followed by α-peptide receptor radionuclide therapy.


Assuntos
Actínio/uso terapêutico , Progressão da Doença , Neoplasias Hepáticas/secundário , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/radioterapia , Octreotida/análogos & derivados , Receptores de Peptídeos/metabolismo , Idoso , Partículas beta/uso terapêutico , Feminino , Humanos , Tumores Neuroendócrinos/metabolismo , Octreotida/uso terapêutico , Resultado do Tratamento
3.
Nat Commun ; 11(1): 333, 2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31949145

RESUMO

Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover metastatic melanoma biology. Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival. We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis. In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters. We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation. Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy.


Assuntos
Perfilação da Expressão Gênica , Melanoma/genética , Melanoma/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Transcriptoma , Animais , Linhagem Celular Tumoral , Retículo Endoplasmático , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Proteína Kangai-1/genética , Proteína Kangai-1/metabolismo , Pulmão/patologia , Melanócitos/metabolismo , Melanoma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica/genética , Segunda Neoplasia Primária/patologia , Fenótipo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Neoplasias Cutâneas/patologia , Ubiquitina-Proteína Ligases/metabolismo
4.
Respir Physiol Neurobiol ; 271: 103310, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31568840

RESUMO

The neuropeptide relaxin-3 is expressed by the pontine nucleus incertus. Relaxin-3 and synthetic agonist peptides modulate arousal and cognitive processes via activation of the relaxin-family peptide 3 receptor (RXFP3). Despite the presence of RXFP3 in the nucleus of the solitary tract (NTS), the ability of RXFP3 to modulate NTS-mediated cardiorespiratory functions has not been explored. Therefore, we examined the effects of bilateral microinjections of the selective agonist, RXFP3-A2 (40 µM, 100 nL/side), into the NTS in perfused working-heart-brainstem-preparations from rats (n = 6), while recording phrenic, vagal, and thoracic sympathetic chain activity (PNA, VNA, t-SCA) and heart rate (HR). RXFP3-A2 significantly increased respiratory rate and shortened post-inspiratory VNA. RXFP3-A2 in the NTS also significantly enhanced arterial chemoreceptor reflex (a-CR)-mediated tachypnea. However, RXFP3-A2 had no significant effect on HR and t-SCA at baseline or during the a-CR. These data represent the first evidence that RXFP3 activation in the NTS can selectively modulate respiration at baseline and during reflex behaviour.


Assuntos
Células Quimiorreceptoras/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Peptídeos/metabolismo , Taxa Respiratória/fisiologia , Núcleo Solitário/metabolismo , Animais , Células Quimiorreceptoras/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Microinjeções/métodos , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/agonistas , Receptores de Peptídeos/agonistas , Taxa Respiratória/efeitos dos fármacos , Núcleo Solitário/efeitos dos fármacos
5.
Sci Adv ; 5(12): eaax0292, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31840061

RESUMO

The mechanistic basis for the biogenesis of peptide hormones and growth factors is poorly understood. Here, we show that the conserved endoplasmic reticulum membrane translocon-associated protein α (TRAPα), also known as signal sequence receptor 1, plays a critical role in the biosynthesis of insulin. Genetic analysis in the nematode Caenorhabditis elegans and biochemical studies in pancreatic ß cells reveal that TRAPα deletion impairs preproinsulin translocation while unexpectedly disrupting distal steps in insulin biogenesis including proinsulin processing and secretion. The association of common intronic single-nucleotide variants in the human TRAPα gene with susceptibility to type 2 diabetes and pancreatic ß cell dysfunction suggests that impairment of preproinsulin translocation and proinsulin trafficking may contribute to the pathogenesis of type 2 diabetes.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Insulina/biossíntese , Glicoproteínas de Membrana/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Estresse do Retículo Endoplasmático , Insulina/metabolismo , Secreção de Insulina , Precursores de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
6.
Sci Adv ; 5(11): eaax3432, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31799390

RESUMO

Can mating influence cognitive functions such as learning and memory in a permanent way? We have addressed this question using a combined behavioral and in vivo imaging approach, finding that aversive long-term memory performance strongly increases in Drosophila females in response to sperm transfer following mating. A peptide in the male sperm, the sex peptide, is known to cause marked changes in female reproductive behavior, as well as other behaviors such as dietary preference. Here, we demonstrate that this sex peptide enhances memory by acting on a single pair of serotonergic brain neurons, in which activation of the sex peptide receptor stimulates the cyclic adenosine monophosphate/protein kinase A pathway. We thus reveal a strong effect of mating on memory via the neuromodulatory action of a sperm peptide on the female brain.


Assuntos
Proteínas de Drosophila/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Memória de Longo Prazo/fisiologia , Receptores de Peptídeos/metabolismo , Neurônios Serotoninérgicos/metabolismo , Espermatozoides/metabolismo , Animais , Encéfalo/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Feminino , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Interferência de RNA , RNA Interferente Pequeno/genética , Comportamento Sexual Animal/fisiologia
7.
Genes (Basel) ; 10(12)2019 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842416

RESUMO

Anti-Mullerian hormone (AMH) is an important reproductive marker of ovarian reserve produced by granulosa cells (GCs) of pre-antral and early-antral ovarian follicles in several species, including cattle. This hormone plays a vital role during the recruitment of primordial follicles and follicle stimulating hormone (FSH)-dependent follicular growth. However, the regulatory mechanism of AMH expression in follicles is still unclear. In this study, we compared the expression of AMH, AMHR-II, BMP2, BMP6, FSHR, and LHCGR genes during follicular development. In-vitro expression study was performed with and without FSH for AMH, AMHR-II, BMP2, and BMP6 genes in bovine GCs which were isolated from 3-8 mm follicles. Association among the mRNA expression and hormone level was estimated. GCs were collected from small (3-8 mm), medium (9-12 mm) and large size (13 to 24 mm) follicles before, during onset, and after deviation, respectively. Further, mRNA expression, hormones (AMH, FSH, and LH), apoptosis of GCs, and cell viability were detected by qRT-PCR, ELISA, flow cytometry, and spectrophotometry. AMH, AMHR-II, BMP2, and FSHR genes were highly expressed in small and medium follicles as compared to large ones. In addition, the highest level of AMH protein (84.14 ± 5.41 ng/mL) was found in medium-size follicles. Lower doses of FSH increased the viability of bovine GCs while higher doses repressed them. In-vitro cultured GCs treated with FSH significantly increased the AMH, AMHR-II, and BMP2 expression levels at lower doses, while expression levels decreased at higher doses. We found an optimum level of FSH (25 ng/mL) which can significantly enhance AMH and BMP2 abundance (p < 0.05). In summary, AMH, AMHR-II, and BMP2 genes showed a higher expression in follicles developed in the presence of FSH. However, lower doses of FSH demonstrated a stimulatory effect on AMH and BMP2 expression, while expression started to decline at the maximum dose. In this study, we have provided a better understanding of the mechanisms regulating AMH, AMHR II, and BMP2 signaling in GCs during folliculogenesis, which would improve the outcomes of conventional assisted reproductive technologies (ARTs), such as superovulation and oestrus synchronization in bovines.


Assuntos
Hormônio Antimülleriano/genética , Bovinos/genética , Células da Granulosa/metabolismo , Animais , Hormônio Antimülleriano/metabolismo , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Estradiol/metabolismo , Feminino , Hormônio Foliculoestimulante/genética , Líquido Folicular/metabolismo , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Folículo Ovariano/metabolismo , RNA Mensageiro/genética , Receptores do FSH/genética , Receptores do FSH/metabolismo , Receptores do LH/genética , Receptores do LH/metabolismo , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo
8.
Elife ; 82019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31710289

RESUMO

Sertoli cells are essential nurse cells in the testis that regulate the process of spermatogenesis and establish the immune-privileged environment of the blood-testis-barrier (BTB). Here, we report the in vitro reprogramming of fibroblasts to human induced Sertoli-like cells (hiSCs). Initially, five transcriptional factors and a gene reporter carrying the AMH promoter were utilized to obtain the hiSCs. We further reduce the number of reprogramming factors to two, NR5A1 and GATA4, and show that these hiSCs have transcriptome profiles and cellular properties that are similar to those of primary human Sertoli cells. Moreover, hiSCs can sustain the viability of spermatogonia cells harvested from mouse seminiferous tubules. hiSCs suppress the proliferation of human T lymphocytes and protect xenotransplanted human cells in mice with normal immune systems. hiSCs also allow us to determine a gene associated with Sertoli cell only syndrome (SCO), CX43, is indeed important in regulating the maturation of Sertoli cells.


Assuntos
Reprogramação Celular , Fibroblastos/metabolismo , Fator de Transcrição GATA4/genética , Síndrome de Células de Sertoli/genética , Células de Sertoli/metabolismo , Fator Esteroidogênico 1/genética , Animais , Diferenciação Celular/genética , Proliferação de Células , Técnicas de Cocultura , Fibroblastos/citologia , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Masculino , Camundongos , Regiões Promotoras Genéticas , Receptores de Peptídeos/genética , Receptores de Peptídeos/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Túbulos Seminíferos/citologia , Túbulos Seminíferos/crescimento & desenvolvimento , Túbulos Seminíferos/metabolismo , Síndrome de Células de Sertoli/metabolismo , Síndrome de Células de Sertoli/patologia , Células de Sertoli/citologia , Células de Sertoli/transplante , Espermatogênese/genética , Espermatogônias/citologia , Espermatogônias/crescimento & desenvolvimento , Espermatogônias/metabolismo , Fator Esteroidogênico 1/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transplante Heterólogo/métodos , Proteínas WT1/genética , Proteínas WT1/metabolismo
9.
Int J Mol Sci ; 20(22)2019 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-31717602

RESUMO

Cellular activities, such as growth and secretion, are dependent on correct protein folding and intracellular protein transport. Injury, like ischemia, malnutrition, and invasion of toxic substances, affect the folding environment in the endoplasmic reticulum (ER). The ER senses this information, following which cells adapt their response to varied situations through the unfolded protein response. Activation of the KDEL receptor, resulting from the secretion from the ER of chaperones containing the KDEL sequence, plays an important role in this adaptation. The KDEL receptor was initially shown to be necessary for the retention of KDEL sequence-containing proteins in the ER. However, it has become clear that the activated KDEL receptor also regulates bidirectional transport between the ER and the Golgi complex, as well as from the Golgi to the secretory pathway. In addition, it has been suggested that the signal for KDEL receptor activation may also affect several other cellular activities. In this review, we discuss KDEL receptor-mediated bidirectional transport and signaling and describe disease models and human diseases related to KDEL receptor dysfunction.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Retículo Endoplasmático/patologia , Estresse do Retículo Endoplasmático , Complexo de Golgi/patologia , Humanos , Transporte Proteico , Proteostase , Receptores de Peptídeos/análise , Transdução de Sinais , Resposta a Proteínas não Dobradas
10.
Results Probl Cell Differ ; 68: 107-125, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31598854

RESUMO

The critical phylogenetic position of the ascidian, Ciona intestinalis, as the closest relative of vertebrates, suggested its potential applicability as a model organism in a wide variety of biological events including the nervous, neuroendocrine, and endocrine regulation. To date, approximately 40 neuropeptides and/or peptide hormones and several cognate receptors have been identified. These peptides are categorized into two types: (1) orthologs of vertebrate peptides, such as cholecystokinin, GnRH, tachykinin, vasopressin, and calcitonin, and (2) novel family peptides such as LF peptides and YFL/V peptides. Ciona GnRH receptors (Ci-GnRHR) were found to be multiplicated in the Ciona-specific lineages and to form unique heterodimers between Ci-GnRHR1 and R4 and between Ci-GnRHR2 and R4, leading to fine-tuning of the generation of second messengers. Furthermore, Ciona tachykinin was shown to regulate a novel protease-associated follicle growth pathway. These findings will pave the way for the exploration of both conserved and diversified endocrine, neuroendocrine, and nervous systems in the evolutionary lineage of invertebrate deuterostomes and/or chordates. In this chapter, we provide an overview of primary sequences, functions, and evolutionary aspects of neuropeptides, peptide hormones, and their receptors in C. intestinalis.


Assuntos
Ciona intestinalis/metabolismo , Neuropeptídeos/metabolismo , Hormônios Peptídicos/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Filogenia
11.
Clin Nucl Med ; 44(11): 876-878, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31584494

RESUMO

Prostate cancer with neuroendocrine differentiation is associated with a poor prognosis, rapid disease progression, and treatment resistance, and constitutes a diagnostic and therapeutic dilemma. We present images of Lu-DOTATATE scan and Ga-DOTATATE PET/CT scan conducted on a 65-year-old man with prostate cancer with neuroendocrine differentiation, whose disease progressed despite conventional treatment and Lu-PSMA radioligand therapy; however, an extraordinary radiographic tumor remission, biochemical response, and improvement of clinical symptoms were observed after the patient underwent Lu-DOTATATE peptide receptor radionuclide therapy.


Assuntos
Dipeptídeos/uso terapêutico , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Tumores Neuroendócrinos/radioterapia , Tumores Neuroendócrinos/secundário , Octreotida/análogos & derivados , Compostos Organometálicos , Neoplasias de Próstata Resistentes à Castração/patologia , Receptores de Peptídeos/metabolismo , Idoso , Humanos , Masculino , Tumores Neuroendócrinos/diagnóstico por imagem , Tumores Neuroendócrinos/metabolismo , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Resultado do Tratamento
12.
Nucl Med Commun ; 40(12): 1195-1203, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31658219

RESUMO

OBJECTIVE: Advanced pancreatic neuroendocrine tumors (pNETs) present a therapeutic challenge with targeted therapies like Everolimus and Lu-DOTATATE peptide receptor radionuclide therapy (PRRT) showing beneficial effects in various cohort studies and randomized trials. Currently there is a paucity of trials with head-to-head comparison between PRRT and Everolimus in advanced pNETs. This systematic review was conducted to compare the therapeutic efficacy and safety profile of Lu-DOTATATE and Everolimus in advanced pNETs. METHODS: The review followed the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guidelines. Searches in Pubmed, Scopus and Embase using relevant keywords selected articles up to June 2019. Data on efficacy and safety were extracted from the individual articles. Random effects model was used for meta-analysis. RESULTS: Fifteen articles consisting of 697 patients reported on Lu-DOTATATE and 12 articles consisting of 946 patients reported on Everolimus. Overall, treatment with Lu-DOTATATE had better objective response rate (47% vs. 12%, P < 0.001) and disease control rate (81% vs. 73%, P < 0.001) and longer progression-free survival (25.7 months vs. 14.7 months, P < 0.001) than with Everolimus. Lu-DOTATATE also had a better safety profile than Everolimus with fewer patients showing grade 3/4 hematological toxicity (5% vs. 11%, P = 0.02) and nephrotoxicity (1% vs. 2.5%, P = 0.34). Treatment-related adverse events caused discontinuation of therapy more frequently for Everolimus than for Lu-DOTATATE (59 out of 371 patients vs. 0 out of 128 patients). CONCLUSION: From this meta-analysis, Lu-DOTATATE showed better therapeutic efficacy and safety profile compared to Everolimus in advanced pNETs.


Assuntos
Complexos de Coordenação/uso terapêutico , Everolimo/uso terapêutico , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/terapia , Octreotida/análogos & derivados , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Receptores de Peptídeos/metabolismo , Humanos , Tumores Neuroendócrinos/metabolismo , Octreotida/uso terapêutico , Neoplasias Pancreáticas/metabolismo
13.
Clin Nucl Med ; 44(12): 989-990, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31663867

RESUMO

Nasopharyngeal carcinoma may express somatostatin receptors (SSTR). We present a case with metastatic nasopharyngeal carcinoma in the liver, bone, and lymph nodes. The patient was in progression after chemotherapy, external beam radiation therapy (ERBT), atezolizumab, and cetuximab. Due to strong SSTR expression of the metastases, PRRT was applied. After 3 cycles of intravenous Lu-DOTATOC and 1 cycle of intraarterial Y-DOTATOC therapy, the hepatic and bone metastases showed excellent response after PRRT. No nephrotoxicity or myelotoxicity was observed.


Assuntos
Carcinoma Nasofaríngeo/patologia , Carcinoma Nasofaríngeo/radioterapia , Octreotida/análogos & derivados , Receptores de Peptídeos/metabolismo , Administração Intravenosa , Adulto , Humanos , Infusões Intra-Arteriais , Masculino , Carcinoma Nasofaríngeo/tratamento farmacológico , Carcinoma Nasofaríngeo/metabolismo , Metástase Neoplásica , Octreotida/administração & dosagem , Octreotida/uso terapêutico , Resultado do Tratamento
14.
J Cell Biol ; 218(10): 3307-3319, 2019 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-31537711

RESUMO

Molecular recognition features (MoRFs) provide interaction motifs in intrinsically disordered protein regions to mediate diverse cellular functions. Here we report that a MoRF element, located in the disordered linker domain of the mammalian signal recognition particle (SRP) receptor and conserved among eukaryotes, plays an essential role in sensing the ribosome during cotranslational protein targeting to the endoplasmic reticulum. Loss of the MoRF in the SRP receptor (SR) largely abolishes the ability of the ribosome to activate SRP-SR assembly and impairs cotranslational protein targeting. These results demonstrate a novel role for MoRF elements and provide a mechanism for the ribosome-induced activation of the mammalian SRP pathway. Kinetic analyses and comparison with the bacterial SRP further suggest that the SR MoRF functionally replaces the essential GNRA tetraloop in the bacterial SRP RNA, providing an example for the replacement of RNA function by proteins during the evolution of ancient ribonucleoprotein particles.


Assuntos
Proteínas de Bactérias/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de Peptídeos/metabolismo , Ribossomos/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Cinética , Transporte Proteico
15.
Ann N Y Acad Sci ; 1456(1): 122-143, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31553068

RESUMO

Latrophilin-1 is an adhesion G protein-coupled receptor that mediates the effect of α-latrotoxin, causing massive release of neurotransmitters from nerve terminals and endocrine cells. Autoproteolysis cleaves latrophilin-1 into two parts: the extracellular N-terminal fragment (NTF) and the heptahelical C-terminal fragment (CTF). NTF and CTF can exist as independent proteins in the plasma membrane, but α-latrotoxin binding to NTF induces their association and G protein-mediated signaling. We demonstrate here that CTF in synapses is phosphorylated on multiple sites. Phosphorylated CTF has a high affinity for NTF and copurifies with it on affinity columns and sucrose density gradients. Dephosphorylated CTF has a lower affinity for NTF and can behave as a separate protein. α-Latrotoxin (and possibly other ligands of latrophilin-1) binds both to the NTF-CTF complex and receptor-like protein tyrosine phosphatase σ, bringing them together. This leads to CTF dephosphorylation and facilitates CTF release from the complex. We propose that ligand-dependent phosphorylation-dephosphorylation of latrophilin-1 could affect the interaction between its fragments and functions as a G protein-coupled receptor.


Assuntos
Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Peptídeos/metabolismo , Animais , Sítios de Ligação , Cromatografia Líquida , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/química , Receptores de Peptídeos/química
16.
Future Oncol ; 15(26): 3015-3024, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31424273

RESUMO

Aim: Data from 69 well-differentiated gastroenteropancreatic neuroendocrine tumors treated with peptide receptor radionucleotide therapy + somatostatin analogs (SSAs) after SSA treatment failure were evaluated. Methods: We identified two groups: S1 - patients who kept the same SSA treatment beyond progression; S2 - patients who switched the SSA with another SSA after progression. Results: Median progression-free survival was 53 and 127 months in S1 and S2, respectively (p = 0.001; hazard ratio: 0.31; 95% CI: 0.15-0.63). Median overall survival was 69 versus 150 months in S1 and S2, respectively (p = 0.004; hazard ratio: 0.32; 95% CI: 0.14-0.71). Conclusion: In patients with advanced well-differentiated gastroenteropancreatic neuroendocrine tumors treated with peptide receptor radionucleotide therapy plus SSA after SSA failure, the 'switch' strategy of SSA after progression improve progression-free survival and overall survival.


Assuntos
Neoplasias Intestinais/patologia , Neoplasias Intestinais/terapia , Marcação por Isótopo , Tumores Neuroendócrinos/patologia , Tumores Neuroendócrinos/terapia , Nucleotídeos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Somatostatina/uso terapêutico , Neoplasias Gástricas/patologia , Neoplasias Gástricas/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Neoplasias Intestinais/metabolismo , Neoplasias Intestinais/mortalidade , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/mortalidade , Nucleotídeos/química , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidade , Prognóstico , Modelos de Riscos Proporcionais , Receptores de Peptídeos/metabolismo , Estudos Retrospectivos , Somatostatina/análogos & derivados , Somatostatina/química , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Resultado do Tratamento
17.
Clin Exp Rheumatol ; 37 Suppl 119(4): 69-75, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31365333

RESUMO

OBJECTIVES: Relaxin is a potent anti-fibrotic hormone that has been tested to ameliorate fibrosis in systemic sclerosis (SSc), but with controversial results. The aim of the study is to sequence relaxin receptor gene RXFP1 and to assess its mRNA expression and protein levels in the skin of SSc patients and healthy subjects. METHODS: Fibroblasts were isolated from unaffected/affected skin samples of (n=16) limited-cutaneous-SSc-(LcSSc) and from affected ones of (n=4) diffuse-cutaneous-SSc-(DcSSc) patients. Fibroblasts from healthy subjects were used as controls. Sequencing of exonic target regions of interest for RXFP1 gene was performed, coupled with mRNA transcript variant analysis. RXFP1 mRNA and protein levels were assessed by quantitative-real-time-PCR-(qRT-PCR) and by immunocytochemistry-(ICC). Alpha-smooth-muscle-actin-(α-SMA) synthesis induced by transforming-growth-factor-beta-1-(TGF-ß1) stimulation was investigated in all fibroblasts with and without pre-treatment with serelaxin (a recombinant form of human relaxin-2 targeting the receptor RXFP1). RESULTS: Sequencing of RXFP1 gene showed no relevant mutations in all fibroblast populations. The analysis of mRNA transcripts revealed the presence of 13 different mRNA isoforms of RXFP1 (7 coding and 6 non-coding) upregulated in LcSSc/DcSSc-affected samples and not in LcSSc-unaffected and in healthy ones. On the contrary, ICC demonstrated the absence of RXFP1 in LcSSc/DcSSc-affected fibroblasts and the presence in LcSSc-unaffected and in healthy ones. To prove these findings, serelaxin pre-incubation was unable to counteract TGF-ß1-driven upregulation of α-SMA in LcSSc/DcSSc-affected fibroblasts only, but not in LcSSc-unaffected and healthy ones. CONCLUSIONS: The absence/altered expression of relaxin receptor RXFP1 in the affected fibroblasts of SSc patients could explain the inefficacy of relaxin-based anti-fibrotic treatments in the disease.


Assuntos
Fibroblastos/metabolismo , Relaxina , Esclerodermia Difusa , Escleroderma Sistêmico , Idoso , Feminino , Fibroblastos/patologia , Fibrose/metabolismo , Fibrose/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Peptídeos/metabolismo , Proteínas Recombinantes , Relaxina/metabolismo , Esclerodermia Difusa/metabolismo , Esclerodermia Difusa/patologia , Escleroderma Sistêmico/metabolismo , Escleroderma Sistêmico/patologia
18.
BMC Cancer ; 19(1): 788, 2019 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395036

RESUMO

BACKGROUND: NETTER-1 trial demonstrated high efficacy and low toxicity of four cycles of Peptide Receptor Radionuclide Therapy (PRRT) in patients with metastasized NET. The present study evaluates the outcome of further PRRT cycles in the so called salvage setting in patients after initial response to four therapy cycles and later progression. METHODS: Thirty five patients (pat.) (25 male, 10 female, 63 ± 9 years) with progressive, metastasized NET (23 small intestinal, 5 lung, 4 CUP, 1 rectal, 1 gastric and 1 paraganglioma) were included. All patients previously received 4 PRRT cycles with 177Lu-DOTATATE and showed initial response. SPECT based dosimetry was applied to determine kidney and tumor doses. Therapy response was evaluated using 68Ga-DOTATATE PET/CT (with high dose CT), CT alone or MRI (RECIST 1.1), toxicity was defined using CTCAE 5.0 criteria. 99mTc99-MAG3 scintigraphy was used to assess potential renal tubular damage. Progression free survival (PFS) and Overall survival (OS) analysis was performed with the Kaplan-Meier-method. RESULTS: The median PFS after initial PRRT was 33 months (95% CI: 30-36). The mean cumulative dose for including salvage PRRT was 44 GBq (range 33.5-47). One pat. (2.9%) showed grade 3 hematotoxicity. Kidney dosimetry revealed a mean cumulative kidney dose after a median of 6 PRRT cycles of 23.8 Gy. No grade 3 / 4 nephrotoxicity or relevant decrease in renal function was observed. Follow-up imaging was available in 32 patients after salvage therapy. Best response according to RECIST 1.1. was PR in one patient (3.1%), SD in 26 patients (81.3%) and PD in 5 patients (15.6%). PFS after salvage therapy was 6 months (95% CI: 0-16; 8 patients censored). Mean OS after initial PRRT was 105 months (95% CI: 92-119) and 51 months (95% CI: 41-61) after start of salvage therapy. Median OS was not reached within a follow-up of 71 months after initial PRRT and 25 months after start of salvage PRRT, respectively. CONCLUSIONS: Salvage therapy with 177Lu-DOTATATE is safe and effective even in patients with extensive previous multimodal therapies during disease progression and represents a feasible and valuable therapy option for progressive NET.


Assuntos
Complexos de Coordenação/uso terapêutico , Tumores Neuroendócrinos/radioterapia , Octreotida/análogos & derivados , Compostos Radiofarmacêuticos/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Complexos de Coordenação/administração & dosagem , Complexos de Coordenação/efeitos adversos , Feminino , Humanos , Estimativa de Kaplan-Meier , Imagem por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/metabolismo , Tumores Neuroendócrinos/mortalidade , Octreotida/administração & dosagem , Octreotida/efeitos adversos , Octreotida/uso terapêutico , Tomografia Computadorizada com Tomografia por Emissão de Pósitrons , Doses de Radiação , Radiometria , Compostos Radiofarmacêuticos/efeitos adversos , Receptores de Peptídeos/metabolismo , Retratamento , Terapia de Salvação , Resultado do Tratamento
19.
Pharmacol Res Perspect ; 7(4): e00513, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31384473

RESUMO

Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein-coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin-dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin-induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine-tagged receptors, using the GABAB heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1-RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin-mediated change in RXFP1-RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT.


Assuntos
Luciferases/metabolismo , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Peptídeos/química , Receptores de Peptídeos/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Células HEK293 , Humanos , Multimerização Proteica , Relaxina/metabolismo , Coloração e Rotulagem
20.
Pestic Biochem Physiol ; 159: 107-117, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400772

RESUMO

Latrophilin (LPH) is an adhesion G protein-coupled receptor (aGPCR) that participates in multiple essential physiological processes. Our previous studies have shown that lph is not only indispensable for the development and reproduction of red flour beetles (Tribolium castaneum), but also for their resistance against dichlorvos or carbofuran insecticides. However, the regulatory mechanism of lph-mediated insecticide susceptibility remains unclear. Here, we revealed that knockdown of lph in beetles resulted in opposing changes in two chemoreception genes, chemosensory protein 10 (CSP10) and odorant-binding protein C01 (OBPC01), in which the expression of TcCSP10 was downregulated, whereas the expression of TcOBPC01 was upregulated. TcCSP10 and TcOBPC01 were expressed at the highest levels in early pupal and late larval stages, respectively. High levels of expression of both these genes were observed in the heads (without antennae) of adults. TcCSP10 and TcOBPC01 were significantly induced by dichlorvos or carbofuran between 12 and 72 h (hrs) after exposure, suggesting that they are likely associated with increasing the binding affinity of insecticides, leading to a decrease in sensitivity to the insecticides. Moreover, once these two genes were knocked down, the susceptibility of the beetles to dichlorvos or carbofuran was enhanced. Additionally, RNA interference (RNAi) targeting of lph followed by exposure to dichlorvos or carbofuran also caused the opposing expression levels of TcCSP10 and TcOBPC01 compared to the expression levels of wild-type larvae treated with insecticides alone. All these results indicate that lph is involved in insecticide susceptibility through positively regulating TcCSP10; and the susceptibility could also further partially compensated for through the negative regulation of TcOBPC01 when lph was knockdown in the red flour beetle. Our studies shed new light on the molecular regulatory mechanisms of lph related to insecticide susceptibility.


Assuntos
Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Receptores de Peptídeos/metabolismo , Tribolium/efeitos dos fármacos , Tribolium/metabolismo , Animais , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Insetos/genética
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